Volume 19, Issue 1, Pages 96-108 (January 2014) β-Aminoisobutyric Acid Induces Browning of White Fat and Hepatic β-Oxidation and Is Inversely Correlated with Cardiometabolic Risk Factors  Lee D. Roberts, Pontus Boström, John F. O’Sullivan, Robert T. Schinzel, Gregory D. Lewis, Andre Dejam, Youn-Kyoung Lee, Melinda J. Palma, Sondra Calhoun, Anastasia Georgiadi, Ming-Huei Chen, Vasan S. Ramachandran, Martin G. Larson, Claude Bouchard, Tuomo Rankinen, Amanda L. Souza, Clary B. Clish, Thomas J. Wang, Jennifer L. Estall, Alexander A. Soukas, Chad A. Cowan, Bruce M. Spiegelman, Robert E. Gerszten  Cell Metabolism  Volume 19, Issue 1, Pages 96-108 (January 2014) DOI: 10.1016/j.cmet.2013.12.003 Copyright © 2014 Elsevier Inc. Terms and Conditions

Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Metabolites Accumulate in the Media of Myocytes as a Result of Forced PGC-1α Expression and Stimulate Expression of Brown Adipocyte-Specific Genes in Adipocytes (A) Media from myocytes transduced with an adenoviral vector expressing either PGC-1α (n = 6) or GFP (n = 6) was analyzed using an LC-MS-based metabolite profiling method. (B) BAIBA (5 μM) induces expression of brown adipocyte-specific genes in primary adipocytes. Additional metabolites tested at physiologically relevant doses included GABA (3 μM), cytosine (1 μM), and 2-deoxycytidine (15 μM). BAIBA significantly increased the expression of the brown adipocyte-specific genes UCP-1 and CIDEA. The expression of adiponectin (ADIPOQ) was unchanged. Cumulative data from a total of five independent observations are shown. (C) BAIBA concentrations in the low micromolar range significantly and dose-dependently increased the expression of the brown adipocyte-specific gene UCP-1. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. Data are represented as mean ± SEM. See also Figure S1. Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 BAIBA Treatment of BJ RiPS Human iPSCs Induces Brown Adipocyte-Specific Gene Expression and Function (A) BAIBA significantly and dose-dependently increased the expression of brown-adipocyte-specific genes in human IPSC-derived mature adipocytes. (B) BAIBA increased both basal and insulin-stimulated glucose uptake in human IPSC-derived adipocytes, assessed by [3H]-2-deoxy-D-glucose transport (data from three independent observations). (C) The oxygen consumption rate (OCR) of human IPSC-derived white adipocytes with and without BAIBA treatment; untransduced cells differentiated with adipogenic media (green line), PPARG2-programmed cells (blue line), and PPARG2-programmed cells treated with BAIBA (black line). The OCR was measured over time with the addition of oligomycin (α), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (β), and antimycin (γ). (D) BAIBA significantly increased the maximal OCR of PPARG2-transduced adipocytes (data are from n = 10 independent observations). (E) Images of untransduced cells, PPARG2-programmed white adipocytes, PPARG2-programmed white adipocytes treated with BAIBA, and PPARG2-CEBPB-programmed brown adipocytes. Shown from left to right: bright-field images illustrating the morphology of the cells; 4′,6-diamidino-2-phenylindole (DAPI) fluorescent nuclear staining (blue), fluorescent staining with the neutral lipid dye BODIPY (green), and fluorescent images of immunostaining with antibodies against the marker protein UCP-1 (red) (100× magnification). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM. See also Figures S2 and S3. Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 BAIBA Induces Expression of Brown Adipocyte-Specific Genes in WAT In Vivo, and Muscle-Specific PGC-1α Expression and Exercise Significantly Increase Plasma BAIBA Levels (A) The plasma concentration of BAIBA in mice given 100 mg/kg/day (n = 5) or 170 mg/kg/day (n = 5) of the metabolite in their drinking water significantly increased over 14 days as compared to age-matched control mice (n = 5). (B) Expression of brown adipocyte-specific genes in inguinal WAT from control mice (−) (n = 5), mice treated with 100 mg/kg/day BAIBA for 14 days (+) (n = 5), or mice treated with 170 mg/kg/day BAIBA for 14 days (++) (n = 5). (C) Plasma BAIBA from muscle-specific PGC-1α transgenic mice (n = 5) was analyzed using LC-MS and compared to age-matched control mice (n = 5). (D) Plasma BAIBA from PGC-1α knockout mice (n = 9) was analyzed using LC-MS and compared to age-matched control mice (n = 8). (E) Mice were subjected to a 3 week free wheel running exercise regimen (n = 6) or housed as sedentary controls (n = 6), and plasma BAIBA levels were assessed by LC-MS. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p < 0.0001. Data are represented as mean ± SEM. Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 BAIBA Decreases Weight Gain and Improves Glucose Tolerance in Mice (A) The weights of mice given 100 mg/kg/day BAIBA (n = 8) in their drinking water compared to untreated controls (n = 8). (B) The percentage body fat of 100 mg/kg/day BAIBA treated mice (n = 8) compared to untreated controls (n = 8). (C) Diurnal oxygen consumption of control mice (n = 8) and BAIBA (100 mg/kg/day)-treated mice (n = 8). (D) Diurnal energy expenditure of control mice (n = 8) and BAIBA (100 mg/kg/day)-treated mice (n = 8). (E) Activity of control mice (n = 8) and mice treated with BAIBA 100 mg/kg/day. (F) Food consumption of control mice and mice treated with BAIBA 100 mg/kg/day. (G) Mice treated with BAIBA 100 mg/kg/day for 14 weeks showed significantly improved glucose tolerance as determined by an IPGTT. (H) The area under the curve of an IPGTT comparing BAIBA-treated mice to untreated controls (control, n = 8; BAIBA, n = 8). ∗p < 0.05. Data are represented as mean ± SEM. Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 PPARα Functions Downstream of BAIBA (A) BAIBA (5 μM) induces expression of PPARα in primary adipocytes over 6 days (six independent observations). (B) Expression of PPARα in inguinal WAT from control mice (n = 5) and mice treated with 100 mg/kg/day BAIBA for 14 days (n = 5). (C) Primary adipocytes treated with BAIBA (5 μM) and/or GW6471 for 6 days. †p < 0.05, ††p < 0.01 compared to BAIBA treatment. (D) BAIBA (5 μM) failed to induce expression of brown adipocyte-specific genes in primary adipocytes isolated from the inguinal WAT of PPARα null mice (six independent observations). (E) Expression of brown adipocyte-specific genes in inguinal WAT from wild-type (WT) control mice (n = 5), WT mice treated with 100 mg/kg/day BAIBA for 14 days (n = 5), PPARα null control mice (n = 5), and PPARα null mice treated with 100 mg/kg/day BAIBA for 14 days (n = 5). ∗p < 0.05, ∗∗p ≤ 0.01 compared to control. Data are represented as mean ± SEM. See also Figure S4. Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 BAIBA Increases Hepatic β-Oxidation through PPARα (A) BAIBA (5 μM) induces expression of fatty acid β-oxidation genes in hepatocytes treated for 6 days (control = 6; BAIBA, 5 μM = 6). (B) BAIBA dose-dependently induces expression of hepatic fatty acid β-oxidation genes in vivo. Expression of fatty acid β-oxidation genes in the liver of control mice (n = 5) and mice treated with 100 mg/kg/day BAIBA for 14 days (n = 5). (C) BAIBA dose-dependently increases the maximal oxygen consumption rate of hepatocytes. (D) qPCR of key β-oxidation genes in hepatocytes treated with BAIBA and/or PPARα antagonist GW6471 for 6 days (data from five independent observations). †††p ≤ 0.001, ††††p < 0.0001 compared to BAIBA treatment. (E) Expression of β-oxidation genes in liver from PPARα null control mice (n = 5) and PPARα null mice treated with 100 mg/kg/day BAIBA for 14 days (n = 5). ∗p < 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p < 0.0001 compared to control. All data are represented as mean ± SEM. Cell Metabolism 2014 19, 96-108DOI: (10.1016/j.cmet.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions